Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate gonadal function and gametogenesis, and are critical for normal sexual development and reproductive function. LH and FSH are synthesized and secreted from pituitary gonadotropes under complex regulation by the hypothalamic peptide, gonadotropin-releasing hormone (GnRH). The long- term objective of this proposal is to gain a better understanding of the molecular mechanisms of the differential regulation of LH and FSH subunit gene expression by GnRH. GnRH is released from the hypothalamus in a pulsatile fashion, with the frequency of release varying throughout the reproductive cycle. Different pulse frequencies regulate differentially LH and FSH biosynthesis and secretion. A major question in reproductive physiology is the mechanism by which this occurs. Previous studies have been limited by the lack of available cell lines which express the LH and FSH subunit genes and respond to GnRH. We have created a novel cell system in which to perform these studies, by transfecting the rat pituitary GH3 cell line with the rat GnRH receptor cDNA. These cells, when cotransfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. The present research proposal focuses on the use of this cell model as an aid to elucidate and compare the mechanisms of regulation of gonadotropin subunit gene expression by GnRH. In particular, this cell model system will be used: (1) to identify the critical cis element(s) in the LHBeta gene which mediates GnRH responses; (2) to identify the critical cis element(s) in the FSHBeta gene which mediates GnRH responses and compare it to that identified in the LHBeta gene; (3) to characterize and compare the trans factors that bind to the cis elements which mediate GnRH responsiveness in the LHBeta and FSHBeta genes; and (4) to confirm the identified mechanisms of GnRH regulation of LHBeta and FSHBeta genes in primary rat pituitary cells, or other gonadotrope cell lines when available. It is expected that these studies will provide new insights into the mechanisms of the regulation of gonadotropins by GnRH, which may in turn provide a better understanding of reproductive development and lead to improved management of disorders of reproductive function.